Recombinant yeast for producing crocin and precursor

Abstract

The present invention relates to a yeast genetically recombined to produce crocin and a precursor thereof and a method for producing crocin and a precursor thereof using same. This invention is the first in the world to successfully establish a biosynthetic pathway for crocin synthesis in yeast. By selecting an enzyme derived from a specific microbial strain for each step of the biosynthetic pathway, the invention achieves significantly superior production yields of crocin and its precursors compared to enzymes derived from other strains. Moreover, the recombinant yeast is highly valuable in terms of its ability to produce all crocin-1, crocin-2, crocin-3, and crocin-4.

Description

Recombinant yeast for producing crocin and its precursors

[0001] The present invention relates to a yeast genetically modified to produce crocin and its precursors and a method for producing crocin and its precursors using the yeast.

[0002] Crocin, a pigmented apocarotenoid, has been widely used in the food industry as a valuable flavoring and coloring agent. Naturally occurring crocin accumulates only in specific tissues of some plants, such as the stigmas of Crocus sativus and the fruits of Gardenia jasminoides. Recently, crocin has been discovered to possess potent pharmacological benefits, including anticancer, anti-inflammatory, and antioxidant properties, as well as potential therapeutic properties against Alzheimer's disease.

[0003] Crocin has two types of glycosyl groups, including D-glucosyl and D-gentiobiosyl, and is divided into crocin-1 (crocetin monoglucosyl ester), crocin-2 (crocetin diglucosyl ester), crocin-3 (crocetin gentiobiosylglucosyl ester), and crocin-4 (crocetin digentiobiosyl ester) depending on the position and number of these two glycosyl groups.

[0004] The stigmas of C. sativus, saffron, are known as the main source of natural crocin and are nicknamed "red gold" due to their complex harvesting process, high labor costs, low yields, and high added value. However, crocin has a complex chemical structure and many chiral center forms, making it difficult to produce through chemical synthesis, and there is a high possibility of forming inactive or toxic optical isomers during the chemical synthesis process. Therefore, the biosynthesis of crocin through microorganisms has recently been the subject of considerable interest and active research. The biosynthetic pathway of crocin, starting from β-carotene, involves four steps catalyzed by β-carotene hydroxylase (CrtZ), carotenoid cleavage dioxygenase (CCD), aldehyde dehydrogenase (ALDH), and UDP-glycosyltransferase (UGT) (Figure 1).

[0005] Saccharomyces cerevisiae is generally considered as a GRAS (Generally Regarded As Safe) strain and has emerged as a major model for terpenoid production due to its many advantages, such as ease of manipulation, high accessibility to genetic information, P450 enzymes, robustness, fast growth rate, high sugar catabolism, and high tolerance to harsh industrial conditions.

[0006] However, to date, no method for biosynthesizing crocin in yeast, including S. cerevisiae, has been known. In particular, no genetically recombinant yeast with a biosynthetic circuit capable of producing all three crocins, crocin-1, crocin-2, crocin-3, and crocin-4, has been known. Therefore, the present inventors aimed to produce crocin using genetically recombinant S. cerevisiae for the first time in the world.

[0007] The present inventors have repeatedly conducted research with the goal of producing crocin by genetically recombinant S. cerevisiae for the first time in the world, and as a result, they have succeeded in constructing an optimal biosynthetic circuit for producing crocin and its precursors by sequentially introducing genes encoding a series of selected specific enzymes into S. cerevisiae, thereby completing the present invention.

[0008] Accordingly, the present invention:

[0009] (a) a gene encoding tHMG1 (truncated HMG-CoA reductase);

[0010] (b) gene encoding CrtE (GGPP synthase);

[0011] (c) a gene encoding CrtYB (bifunctional phytoene synthase and lycopene cyclase); and

[0012] (d) The purpose is to provide a recombinant yeast for producing crocin and its precursors, into which a gene encoding CrtI (phytoene desaturase) is introduced.

[0013] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin and its precursor, into which a gene encoding (e) CrtZ (β-carotene hydroxylase) is additionally introduced.

[0014] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin and its precursor, into which a gene encoding (f) CCD (carotenoid cleavage dioxygenase) is additionally introduced.

[0015] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin and its precursor, into which a gene encoding (g) ALDH (aldehyde dehydrogenase) is additionally introduced.

[0016] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin and its precursor, into which a gene encoding NtUGT (UDP-glycosyltransferase) derived from (h) Nicotiana tabacum is additionally introduced.

[0017] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin and its precursor, into which (i) a gene encoding CaUGT3 (UDP-glycosyltransferase, UDP-glycosyltransferase) derived from Catharanthus roseus has been additionally introduced.

[0018] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin-1 and crocin-2, in which a gene encoding NtUGT derived from Nicotiana tabacum is introduced into a yeast genetically modified to produce crocetin.

[0019] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin-3 and crocin-4, in which a gene encoding NtUGT derived from Nicotiana tabacum and a gene encoding CaUGT3 derived from Catharanthus roseus are introduced into a yeast genetically recombinant to produce crocetin.

[0020] In addition, another object of the present invention is to provide a recombinant yeast for producing crocin-3 and crocin-4, in which a gene encoding CaUGT3 derived from Catharanthus roseus is introduced into a yeast genetically recombinant to produce crocin-2.

[0021] In addition, another object of the present invention is to provide a method for producing crocin and its precursor, which comprises a step of culturing the recombinant yeast.

[0022] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0023] In order to achieve the above-described purpose of the present invention, in one aspect, the present invention:

[0024] (a) gene encoding tHMG1;

[0025] (b) gene encoding CrtE;

[0026] (c) a gene encoding CrtYB; and

[0027] (d) Provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding CrtI is introduced.

[0028] In one embodiment, the recombinant yeast may produce β-carotene, a precursor of crocin.

[0029] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (e) CrtZ is additionally introduced.

[0030] In one embodiment, the CrtZ gene may be derived from Pantoea ananatis.

[0031] In one embodiment, the recombinant yeast may produce zeaxanthin, a precursor of crocin.

[0032] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (f) CCD is additionally introduced.

[0033] In one embodiment, the CCD gene may be derived from Crocus Sativus.

[0034] In one embodiment, the recombinant yeast may produce crocetin dialdehyde, a precursor of crocin.

[0035] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (g) ALDH is additionally introduced.

[0036] In one embodiment, the ALDH gene may be derived from Crocus Sativus.

[0037] In one embodiment, the recombinant yeast may produce crocetin, a precursor of crocin.

[0038] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding NtUGT derived from (h) Nicotiana tabacum is additionally introduced.

[0039] In one embodiment, the recombinant yeast may produce crocin-1 and crocin-2.

[0040] In another aspect, the present invention provides (i) a recombinant yeast for producing crocin and its precursors, into which a gene encoding CaUGT3 derived from Catharanthus roseus is additionally introduced.

[0041] In one embodiment, the recombinant yeast may produce crocin-3 and crocin-4.

[0042] In another aspect, the present invention provides a recombinant yeast for producing crocin-1 and crocin-2, wherein a gene encoding NtUGT from Nicotiana tabacum is introduced into a yeast genetically modified to produce crocetin.

[0043] In another aspect, the present invention provides a recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding NtUGT from Nicotiana tabacum and a gene encoding CaUGT3 from Catharanthus roseus are introduced into a genetically modified yeast for producing crocetin.

[0044] In another aspect, the present invention provides a recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding CaUGT3 derived from Catharanthus roseus is introduced into a yeast genetically modified to produce crocin-2.

[0045] In one embodiment, the yeast in all of the above aspects may be Saccharomyces cerevisiae.

[0046] In another aspect, the present invention provides a method for producing crocin and its precursor, comprising the step of culturing the recombinant yeast.

[0047] The present invention relates to a yeast genetically recombinant to produce crocin and its precursors, and a method for producing crocin and its precursors using the same. The present invention is significant in that it succeeded in constructing a biosynthetic circuit for synthesizing crocin in yeast for the first time in the world, and by selecting enzymes derived from specific strains for each step of the biosynthetic circuit, the production of crocin and its precursors is significantly superior to enzymes derived from other strains, and is very useful in that it can produce all of crocin-1, crocin-2, crocin-3, and crocin-4.

[0048] Figure 1 shows the crocin biosynthetic pathway of recombinant Saccharomyces cerevisiae. The related genes are as follows: tHMG1 (truncated HMG-CoA reductase); CrtE (GGPP synthase); CrtYB (bifunctional phytoene synthase and lycopene cyclase); CrtI (phytoene desaturase); CrtZ (β-carotene hydroxylase); CCD (carotenoid cleavage dioxygenase); ALDH (aldehyde dehydrogenase); UGT (UDP-glycosyltransferase).

[0049] Figure 2 illustrates the construction of the zeaxanthin pathway and confirmation of zeaxanthin production in Saccharomyces cerevisiae. (A) Schematic diagram of an engineered strain expressing the CrtZ enzyme for zeaxanthin biosynthesis. (B) HPLC analysis of zeaxanthin extracts and zeaxanthin standards from zeaxanthin-producing strains with three CrtZ sources. Peak 1: zeaxanthin, Peak 2: beta-carotene. (C) UV / Vis spectral analysis of compounds corresponding to peaks 1 and 2 in the HPLC chromatogram. (D) Quantitative analysis of zeaxanthin according to CrtZ sources.

[0050] Figure 3 shows the confirmation of the physiological activity of CsCCD2. (A) Schematic diagram of the CCD enzyme expression cassette on the S. cerevisiae chromosome. (B) HPLC analysis of extracts of a recombinant strain expressing the CCD enzyme and standards for crocetin dialdehyde and crocetin. Peak 1: crocetin, Peak 2: crocetin dialdehyde, and Peak 3: zeaxanthin. (C) UV / Vis spectral analysis of compounds corresponding to Peaks 1-3 in the HPLC chromatogram. (D) Transcript levels of ccd2 were analyzed by RT-PCR. The pma1 gene was used as a reference gene.

[0051] Figure 4 illustrates a comparison of ALDH for crocetin production. (A) Schematic illustration of a crocetin-producing strain containing the pRS424-aldh plasmid. (B) HPLC analysis of extracts from the crocetin-producing strain and a crocetin standard. Peak 1: crocetin, Peak 2: zeaxanthin. (C) UV / Vis spectral analysis of compounds corresponding to peaks 1 and 2 in the HPLC chromatogram. (D) Production of zeaxanthin and crocetin in a crocetin-producing strain.

[0052] Figure 5 shows a recombinant crocin-2-producing strain. (A) Strain and plasmid construction diagram constructed to produce crocin-1 and 2 in S. cerevisiae. (B) HPLC analysis of the medium and cell pellet of the crocin-2-producing strain. Peak 1: crocin-2, Peak 2: crocin-1, Peak 3: crocetin. (C) UV / Vis spectral analysis of compounds corresponding to peaks 1-3 in the HPLC chromatogram. (D) Production of crocetin, crocin-1, and crocin-2.

[0053] Figure 6 shows a recombinant crocin-4 producing strain. (A) Strain and plasmid construction diagram constructed to produce crocin-3 and 4 in S. cerevisiae. (B) HPLC analysis of medium extracts of the crocin-4 producing strain. Peak 1: crocin-4, Peak 2: crocin-3, Peak 3: crocin-2. (C) UV / Vis spectral analysis of compounds corresponding to peaks 1-3 in the HPLC chromatogram. (D) Production of crocin-2, crocin-3, and crocin-4.

[0054] Figure 7 illustrates the reconstruction of the crocetin production pathway in yeast (S. cerevisiae) and confirmation of crocetin production. (A) Schematic diagram of an engineered strain expressing the CsALDH enzyme for crocetin biosynthesis. (B) HPLC analysis peaks of crocetin and zeaxanthin in cell pellet extracts from the crocetin-producing strain CEN-ZPECA. (C) UV / Vis spectral analysis of compounds corresponding to peaks in the HPLC chromatogram.

[0055] Figure 8 shows the reconstruction of the crocin-2 production pathway in S. cerevisiae and confirmation of crocin-2 production. (A) Schematic diagram of an engineered strain expressing the GjUGT1 enzyme for crocin-2 biosynthesis. (B) HPLC analysis peaks of crocin-2 and zeaxanthin in the cell pellet and medium from the crocin-2-producing CEN-ZPECAG strain. (C) UV / Vis spectral analysis of compounds corresponding to peaks in the HPLC chromatogram of the CEN-ZPECAG strain. (D) Schematic diagram of an engineered strain expressing the NtUGT enzyme for crocin-2 biosynthesis. (E) HPLC analysis peaks of crocin-2 and zeaxanthin in the cell pellet and medium from the crocin-2-producing CEN-ZPECAN strain. (F) UV / Vis spectral analysis of compounds corresponding to peaks in the HPLC chromatogram of the CEN-ZPCAN strain.

[0056] Figure 9 illustrates the reconstruction of the crocin-4 production pathway in S. cerevisiae and confirmation of crocin-4 production. (A) Schematic illustration of the crocin-2-producing strain CEN_ZPECAN containing the pRS424-CaUGT3 plasmid. (B) HPLC analysis peaks for the medium extract of the crocin-4-producing strain. (C) Peaks in the HPLC chromatogram correspond to UV / Vis spectral analysis of compounds corresponding to crocin-2 and crocin-4.

[0057] To construct a biosynthetic circuit for synthesizing crocin in yeast, the inventors first constructed a beta-carotene pathway.

[0058] Therefore, in one aspect, the present invention:

[0059] (a) gene encoding tHMG1;

[0060] (b) gene encoding CrtE;

[0061] (c) a gene encoding CrtYB; and

[0062] (d) Provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding CrtI is introduced.

[0063] To help understand the present invention, the crocin production pathway of the present invention is shown in the following reaction scheme 1.

[0064] [Reaction Formula 1]

[0065]

[0066] With reference to the above reaction scheme 1, the terms used herein are defined as follows.

[0067] The term "crocin" used herein refers to the final products of the above reaction scheme 1, which are divided into crocin-1 (crocetin monoglucosyl ester), crocin-2 (crocetin diglucosyl ester), crocin-3 (crocetin gentiobiosylglucosyl ester), and crocin-4 (crocetin digentiobiosyl ester) depending on the positions and numbers of D-glucosyl and D-gentiobiosyl. Therefore, "crocin" herein may comprehensively refer to crocin-1 to crocin-4.

[0068] The term "(crocin) precursor" as used herein may refer to beta-carotene, zeaxanthin, crocetin dialdehyde and crocetin as intermediates for synthesizing crocin.

[0069] The term "tHMG1" as used herein is an abbreviation for truncated HMG-CoA reductase, which is an enzyme that converts HMG-CoA to MVA (Mevalonate). In one embodiment, the tHMG1 gene may be, but is not limited to, the HMG1 gene derived from the S. cerevisiae strain, CEN.PK2-1D. Specifically, the HMG1 gene may encode amino acids 553-1054 of the HMG1 protein, but is not limited thereto.

[0070] MVA is converted to geranyl diphosphate (GPP) via the MVA pathway, and GPP is then converted to farnesyl diphosphate (FPP).

[0071] The term "CrtE" used herein refers to GGPP synthase, an enzyme that acts on FPP to synthesize GGPP.

[0072] The term "CrtYB" as used herein refers to bifunctional phytoene synthase and lycopene cyclase, which is an enzyme that acts on GGPP to build a backbone to synthesize phytoene and cyclizes both ends of unsaturated lycopene to form beta-carotene.

[0073] The term "CrtI" used herein refers to phytoene desaturase, an enzyme that acts on phytoene to desaturate single bonds and form lycopene. Lycopene is cyclized by CrtYB to form beta-carotene.

[0074] In one embodiment, the CrtE, CrtYB and CrtI genes may be derived from, but are not limited to, a Xanthophyllomyces dendrorhous strain.

[0075] Thus, recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; and (d) a gene encoding CrtI have been introduced can ultimately produce beta-carotene.

[0076] In the examples described below, the beta-carotene pathway was constructed using CEN.PK2-1D, a S. cerevisiae experimental strain as a yeast, and the finally constructed strain was named CEN-BetaY (CEN.PK2 1-D Δ his3 :: tHMG1 Δ ypl062w :: CrtE Δ rox1 :: CrtYB Δ yjl064w :: CrtI).

[0077] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (e) CrtZ is additionally introduced.

[0078] The term "CrtZ" used herein refers to β-carotene hydroxylase, an enzyme that synthesizes zeaxanthin by adding hydroxyl groups to the rings at both ends of β-carotene.

[0079] The present inventors selected heterologous CrtZ enzymes of different origins (from Flavobacterium kingsejongi, Flavobacterium fecale, and Pantoea ananatis) as candidates for converting beta-carotene to zeaxanthin. Among these, the CrtZ enzyme from Pantoea ananatis showed the highest zeaxanthin activity in terms of zeaxanthin production and beta-carotene accumulation, and was ultimately selected.

[0080] Thus, in one embodiment, the CrtZ gene may be derived from, but is not limited to, Pantoea ananatis.

[0081] Thus, a recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; and (e) a gene encoding CrtZ has been introduced can ultimately produce zeaxanthin.

[0082] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (f) CCD is additionally introduced.

[0083] The term "CCD" as used herein refers to carotenoid cleavage dioxygenase, an enzyme that cleaves the rings at both ends of zeaxanthin and combines two oxygen atoms to synthesize crocetin dialdehyde.

[0084] In one embodiment, the CCD gene may be derived from, but is not limited to, Crocus Sativus.

[0085] Thus, a recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; (e) a gene encoding CrtZ; and (f) a gene encoding CCD has been introduced can ultimately produce crocetin dialdehyde.

[0086] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding (g) ALDH is additionally introduced.

[0087] The term "ALDH" used herein refers to aldehyde dehydrogenase, an enzyme that acts on crocetin dialdehyde to oxidize it into crocetin.

[0088] The present inventors selected two types of aldehyde dehydrogenases, CsALDH3I1 from Crocus sativus and synALDH7942 from Synechococcus elongatus PCC7942, as candidates, and conducted experiments to select a more suitable enzyme when creating a crocetin-producing strain using S. cerevisiae as a model. As a result, the CsALDH3I1 enzyme from Crocus sativus showed higher crocetin activity in terms of crocetin production and zeaxanthin accumulation, and was ultimately selected.

[0089] Thus, in one embodiment, the ALDH gene may be derived from, but is not limited to, Crocus Sativus.

[0090] Thus, a recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; and (g) a gene encoding ALDH has been introduced can ultimately produce crocetin.

[0091] In another aspect, the present invention provides a recombinant yeast for producing crocin and its precursors, into which a gene encoding NtUGT derived from (h) Nicotiana tabacum is additionally introduced.

[0092] The term "UGT" as used herein refers to UDP-glycosyltransferase, and "NtUGT" refers to UGT derived from Nicotiana tabacum. UGT is an enzyme that acts on crocetin to synthesize crocin-1 and crocin-2.

[0093] NtUGT derived from Nicotiana tabacum is a novel enzyme discovered by the present inventors and is described in Korean Patent No. 10-2343323, which describes the production of crocin-1 and crocin-2 from crocetin using this enzyme in E. coli.

[0094] In the examples described below, the inventors of the present invention compared the previously known GjUGT1 enzyme derived from Gardenia jasminoides with the NtUGT enzyme, and as a result, confirmed that the conversion to crocin was superior when NtUGT was used, and thus the NtUGT enzyme was ultimately selected.

[0095] Thus, recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; (g) a gene encoding ALDH; and (h) a gene encoding NtUGT have been introduced can ultimately produce crocin-1 and crocin-2.

[0096] In another aspect, the present invention provides (i) a recombinant yeast for producing crocin and its precursors, into which a gene encoding CaUGT3 derived from Catharanthus roseus is additionally introduced.

[0097] The term "UGT" as used herein refers to UDP-glycosyltransferase, and "CaUGT3" refers to UGT derived from Catharanthus roseus. CaUGT3 is an enzyme that acts on crocin-2 to synthesize crocin-3 and crocin-4.

[0098] CaUGT3 derived from Catharanthus roseus is a novel enzyme discovered by the present inventors and is described in Korean Patent No. 10-2335035, which describes the production of crocin-3 and crocin-4 from crocetin using this enzyme in E. coli.

[0099] Thus, recombinant yeast into which (a) a gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; (g) a gene encoding ALDH; (h) a gene encoding NtUGT; and (i) a gene encoding CaUGT3 have been introduced can ultimately produce crocin-3 and crocin-4.

[0100] In another aspect, the present invention provides a recombinant yeast for producing crocin-1 and crocin-2, wherein a gene encoding NtUGT from Nicotiana tabacum is introduced into a yeast genetically modified to produce crocetin.

[0101] Here, the yeast genetically recombined to produce the above crocetin may be a recombinant yeast into which the genes (a) to (g) described above have been introduced, but is not necessarily limited thereto, and may be a recombinant yeast genetically recombined in another way to produce crocetin.

[0102] In another aspect, the present invention provides a recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding NtUGT from Nicotiana tabacum and a gene encoding CaUGT3 from Catharanthus roseus are introduced into a genetically modified yeast for producing crocetin.

[0103] Likewise, the yeast genetically recombined to produce the above crocetin may be a recombinant yeast into which the genes (a) to (g) described above have been introduced, but is not necessarily limited thereto, and may be a recombinant yeast genetically recombined in another way to produce crocetin.

[0104] In another aspect, the present invention provides a recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding CaUGT3 derived from Catharanthus roseus is introduced into a yeast genetically modified to produce crocin-2.

[0105] Here, the yeast genetically recombined to produce the above crocin-2 may be a recombinant yeast into which the genes (a) to (h) described above have been introduced, but is not necessarily limited thereto, and may be a recombinant yeast genetically recombined in another way to produce crocin-2.

[0106] In all of the above aspects, the yeast may be a commonly used microorganism of the genus Saccharomyces. Specifically, the microorganism of the genus Saccharomyces may be Saccharomyces cerevisiae.

[0107] In one embodiment of the present invention, the yeast may be, but is not limited to, Saccharomyces cerevisiae. Specifically, the S. cerevisiae may be, but is not limited to, the experimental strain, CEN.PK2-1D.

[0108] The term "recombinant yeast" as used herein refers to yeast transformed with one or more genes from (a) to (i) introduced therein. Here, introduction of the gene includes not only direct integration of the gene into the yeast chromosome, but also co-expression of the gene via a vector such as a plasmid.

[0109] The recombinant yeast described above can use a vector to remove or introduce a gene, and the type of the vector is not particularly limited, and can be produced using any vector known in the art. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. The vectors that can be used in the present invention are not particularly limited, and any known expression vector can be used.

[0110] In another aspect, the present invention provides a method for producing crocin and its precursor, comprising the step of culturing the recombinant yeast.

[0111] Here, the description of the recombinant yeast is as described above.

[0112] The term "culture" used herein refers to the growth of target cells or tissues under artificially controllable environmental conditions. Artificially controlled environmental conditions typically include nutrients, temperature, osmotic pressure, pH, gas composition, and light. However, the medium directly influences the growth, which is broadly divided into liquid and solid media.

[0113] In one embodiment, the production method may further comprise a step of recovering crocin and its precursors from the cultured yeast or a culture thereof.

[0114] The step of recovering crocin and its precursors can be performed by any suitable method known in the art, depending on the culture method. Specifically, the recovery method is not particularly limited thereto, but may include, but is not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., HPLC, ion exchange, affinity, hydrophobicity, and size exclusion), and the like.

[0115] The significance of the genetically recombinant yeast of the present invention and the method for producing crocin and its precursors using the same lies in the fact that it succeeded in constructing a biosynthetic circuit for synthesizing crocin in yeast for the first time in the world, and by selecting enzymes derived from specific strains for each step of the biosynthetic circuit, the production of crocin and its precursors is significantly superior to enzymes derived from other strains, and is very useful in that it can produce all of crocin-1, crocin-2, crocin-3, and crocin-4.

[0116]

[0117] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. However, these examples are intended only to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0118]

[0119] Example

[0120] Example 1: Implementation method

[0121] 1.1. Strains and culture conditions

[0122] Escherichia coli XL1-Blue was used for plasmid construction by cloning the genes of the crocin biosynthetic pathway and was cultured in LB (0.5% yeast extract, 1% tryptone, and 0.5% NaCl) medium. 30 μg / mL kanamycin, 100 μg / mL ampicillin, or 50 μg / mL chloramphenicol antibiotics were supplemented for selection as needed. Saccharomyces cerevisiae, CEN.PK2-1D (MATα) strain was used to construct the crocin biosynthetic pathway. YPD medium (2% peptone, 1% yeast extract, 2% glucose) was used for genome editing, and after transformation, selection was performed on SD plates (0.67% amino acid-free yeast nitrogen base, 2% glucose, and the appropriate amino acid drop-out mix) or YPD plates containing 50 μg / mL nortricin. For carotenoid production, engineered recombinant strains were cultured in SD medium with appropriate amino acid drop-out mix.

[0123]

[0124] 1.2. Construction of plasmids for expression of enzymes of the crocin biosynthetic pathway

[0125] The strains, plasmids, and PCR primers used in this study are listed in Tables 1 and 2.

[0126] The CrtYB (bifunctional phytoene synthase and lycopene cyclase), CrtI (phytoene desaturase), and CrtE (GGPP synthase) genes used to construct the yeast beta-carotene production pathway were derived from Xanthophyllomyces dendrorhous strains. The CrtYB, CrtI, and CrtE genes were amplified by conventional PCR methods and cloned into the pRS series plasmids or pUC57-URA3 plasmids containing a strong constitutive expression promoter. The tHMG1 gene was amplified from the HMG1 gene (HMG-CoA reductase) of the S. cerevisiae strain, CEN.PK2-1D. The resulting tHMG1 gene (truncated HMG-CoA reductase) encodes amino acids 553-1054 of the HMG1 protein. The amplified PCR fragment of the tHMG1 gene was cloned into the pRS426 plasmid and the pUC57-URA3 plasmid, which have strong constitutive expression promoters. Next, to construct a crocin-producing strain, CrtZ (β-carotene hydroxylase) from Flavobacterium kingsejongi, Flavobacterium faecale, and Pantoea ananatis was cloned into the BamHI and SalI restriction enzyme sites of the pRS425 plasmid and expressed under the strong constitutive expression promoter GPD. CsCCD2 from Crocus sativus, synALDH7942 from Synechococcus elongatus PCC 7942, and CsALDH3I1 from Crocus sativus were all synthesized as S. cerevisiae codon-optimized versions. The synthetic genes were amplified by a conventional PCR method using corresponding primers. The PCR fragments were inserted into the pRS series plasmids or the YIPlac128 plasmid.

[0127] To construct plasmids related to the UDP-glycosyltransferase gene, the plasmids pUCrop_GjUGT1 and pUCrop_NtUGT_CaUGT3 used in the inventors' laboratory were used. The GjUGT1 (UGT75L6) gene from Gardenia jasminoides, the NtUGT gene from Nicotiana tabacum, and the CaUGT3 gene from Catharanthus roseus were each amplified by a general PCR method and cloned into the pRS series plasmid.

[0128] The Gibson cloning method was used to express two to three expression modules in a single plasmid. CaUGT3 was amplified from pRS424_CaUGT3, and the PCR fragment was inserted into the pRS426_NtUGT plasmid to construct pRS426_NtUGT_CaUGT3.

[0129] All ligation mixtures were directly transformed into E. coli XL1-Blue using electrophoretic transformation. Successful transformants were selected on Luria-Bertani broth (LB) plates containing 100 µg / ml ampicillin.

[0130]

[0131] 1.3. Construction of β-carotene-producing strains

[0132] All plasmids or linear DNAs were transformed using the LiAc / SS carrier DNA / PEG method.

[0133] The beta-carotene pathway was constructed using the S. cerevisiae experimental strain, CEN.PK2-1D. The expression cassettes for tHMG1 and crtE were integrated into the chromosome using the URA blaster method. First, the PPGK1-tHMG1-TCYC1-URA3 replacement cassette, which has 50 bp of homology to the his3 gene on both sides, was amplified from the pUC57-URA3-tHMG1 plasmid and introduced into S. cerevisiae (CEN.PK2 1-D Δ his3 :: tHMG1). Subsequently, the PTEF1-CrtE-TCYC1-URA3 cassette was integrated into the ypl062w gene region using the same method to construct the tHMG1 / CrtE strain (CEN.PK2 1-D Δ his3 :: tHMG1 Δ ypl062w :: CrtE ). All strains generated by the URA blaster method were selected on SD(URA-) plates and also counterselected on 5-fluoroorotic acid (FOA) plates, resulting in the removal of all URA3 blaster cassettes.

[0134] Next, integration of the CrtYB and CrtI genes in S. cerevisiae was performed using the CRISPR-Cas9 method. Two guide RNA expression vectors targeting rox1 and yjl064w, respectively, were designed and constructed using the CHOP CHOP program (https: / / chopchop.cbu.uib.no / ). The pCas9-NAT and pSNR52_URA3_ROX1 plasmids and the PPGK1-CrtYB-TCYC1 cassette were inserted into the tHMG1 / CrtE strain to construct the tHMG1 / CrtE / CrtYB strain (CEN.PK2 1-D Δ his3 :: tHMG1 Δ ypl062w :: CrtE Δ rox1 :: CrtYB). Next, the PPGK1-CrtI-TADH1 replacement cassette was integrated into the yjl064w gene locus with the help of pCas9-NAT and pSNR52_URA3_YJL064W. The strain engineered using the CRISPR-Cas9 system was selected on SD (URA-, NAT) plates and counterselected on 5-FOA plates.

[0135] The finally constructed strain was named CEN-BetaY (CEN.PK2 1-D Δ his3 :: tHMG1 Δ ypl062w :: CrtE Δ rox1 :: CrtYB Δ yjl064w :: CrtI). At each stage of strain construction, PCR was performed using primer pairs adjacent to the ends of each target gene region, and the mutations in each target region were confirmed through sequencing.

[0136]

[0137] 1.4. Integration of CrtZ and CCD genes into the chromosome

[0138] Transformation of S. cerevisiae strains was performed using the conventional lithium acetate method. The PGPD-CrtZ-TCYC1 cassette containing the HIS3 marker was integrated into the genome of strain CEN-BetaY. The method is as follows. To construct the integrated cassette PGPD-CrtZ-TCYC1-HIS3, primers were designed so that the linear DNAs of PGPD-CrtZ-TCYC1 and the HIS3 expression module could overlap each other. Using the designed primers, each module was amplified from pRS425_PGPD-CrtZ-TCYC1 and pRS423. Next, each fragment was joined using primers containing the LPP1 gene and a 50-bp homologous recombination region and amplified by the OE-PCR method. Transformants introducing the amplified PGPD-CrtZ-TCYC1-HIS3 cassette into the CEN-BetaY strain were selected on SD(-HIS) plates, screened by colony PCR, and finally confirmed by sequencing. Depending on the origin of the CrtZ gene, strains CEN-ZP, CEN-ZFf, and CEN-ZFk were obtained.

[0139] Next, to integrate the PPGK1-CsCCD2-TTEF2 expression module into the CEN-ZP strain, the expression module was subcloned from pRS424_PPGK1-CCD-TTEF2 into the YIplac128 integrative plasmid containing the LEU2 marker. The PPGK1-CCD-TTEF2-LEU2 replacement cassette was amplified from YIplac128_PPGK1-CCD-TTEF2-LEU2 using primers containing the TRP1 gene and a 50-bp homologous recombination region. Transformants were selected on SD(-LEU) plates, and strains were selected by colony PCR analysis and confirmed by sequencing, obtaining strain CEN-ZPC.

[0140]

[0141] 1.5. Reverse transcription PCR (RT-PCR)

[0142] Total RNA was extracted from S. cerevisiae cells using the easy BLUETM Total RNA Extraction Kit (Intron, Seoul, South Korea). For reverse transcription polymerase chain reaction (RT-PCR) analysis, cDNA was synthesized from total RNA samples using the ReverTraTMAce qPCR RT Kit (Toyobo, Osaka, Japan). RT-PCR conditions were as follows: denaturation at 95°C for 1 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. The gene PMA1, encoding a plasma membrane ATPase, was used as a reference gene.

[0143]

[0144] 1.6. Carotenoid extraction and analysis

[0145] All solvents used in chromatographic analyses were of HPLC grade and purchased from SAMCHUN. To extract beta-carotene, zeaxanthin, crocetin, and crocin-1, cells were harvested by high-speed centrifugation (4,000 rpm, 4°C, 20 min). After removing the supernatant, the cell pellet was resuspended in MeOH:Acetone (50:50, v / v) and crushed with glass beads using a bead beater. To extract crocetin, crocin-1, crocin-2, crocin-3, and crocin-4, the culture medium was lyophilized using a freeze-dryer and resuspended in MeOH. All organic solvent phases were filtered through a 0.2 μm PTFE syringe membrane filter and dried using a Genevac EZ-2 centrifugal evaporator (Fisher Scientific, UK). The dried pellet was dissolved in 100 μL of MeOH or MeOH: Dimethylformamide (7:1, v / v), and 10 μL was analyzed using a high-performance liquid chromatography (1200 series; Agilent Technology, USA) equipped with a UV / VIS detector (435 nm; Agilent Technology) and a ZORBAX Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm, Agilent Technology). Mobile phase A (MeOH) and mobile phase B (DDW) were used for gradient elution at 0.8 mL / min as follows: Mobile phase A: 50-80% (0-60 min), 80-100% (60-80 min), 100-50% (80-90 min), 50% (90-100 min), and the column temperature was maintained at 40°C.

[0146] Table 1. Strains and plasmids used in this study

[0147]

[0148]

[0149]

[0150] Table 2. Oligonucleotides used in this study

[0151]

[0152]

[0153] Example 2: Results

[0154] 2.1. Construction of the zeaxanthin biosynthetic pathway in S. cerevisiae

[0155] The beta-carotene-producing strain CEN-BetaY was constructed using the CrtE, CrtYB, and CrtI genes from Xanthyllomyces dendrorhous along with overexpression of a truncated HMG1. CEN-BetaY was used as a parent strain for integration of the CrtZ expression module encoding beta-carotene hydroxylase. Heterologous CrtZs from different origins (from Flavobacterium kingsejongi, Flavobacterium fecale, and Pantoea ananatis) were selected as candidates for converting beta-carotene to zeaxanthin. Each CrtZ gene was integrated into the CEN-BetaY chromosome to construct strains CEN-ZFk, CEN-ZFF, and CEN-ZP (Fig. 2A). To find the optimal CrtZ enzyme, the zeaxanthin-producing strains were cultured in 50 ml SD medium. After cultivation, the extracts were analyzed using HPLC-DAD. Extracts from all three strains engineered with the CrtZ expression module detected peaks with retention times identical to those of the zeaxanthin standard, and all exhibited UV / Vis spectra identical to those of the standard (Figs. 2B and 2C). Zeaxanthin production and beta-carotene accumulation were the criteria for selecting CrtZ. Therefore, strain CEN_ZP was ultimately selected based on the titers (mg / L) of zeaxanthin and beta-carotene (Fig. 2D).

[0156]

[0157] 2.2. Confirmation of CsCCD2 physiological activity

[0158] Next, to integrate the CsCCD2 gene from Crocus Sativus into the CEN-ZP strain, the corresponding expression cassette was cloned into YIplac128 to construct YIplac_pPGK1_CsCCD2_tTEF2 (Table 1). After constructing the strain CEN-ZPC (Fig. 3A), the extract of CEN-ZPC grown in a 50 mL flask was analyzed together with zeaxanthin, crocetin dialdehyde, and a crocetin standard. As a result, one new peak (peak 1 in Fig. 3B) was detected in the extract of CEN-ZPC at the same retention time as the crocetin standard with the same UV / Vis spectrum (Fig. 3C). However, no peak corresponding to crocetin dialdehyde was detected. This suggests that crocetin dialdehyde produced by CsCCD2 is directly converted to crocetin by ALDH, which is endogenously expressed in S. cerevisiae. The expression of CsCCD2 was indirectly confirmed by RT-PCR (Fig. 3D). In summary, the CsCCD2 gene was functionally expressed in the CEN-ZPC strain and produced crocetin, confirming its physiological activity.

[0159]

[0160] 2.3. Comparison of ALDH for crocetin production

[0161] As previously reported in overseas journals, CsALDH3I1 derived from Crocus sativus was verified to oxidize crocetin dialdehyde to crocetin in S. cerevisiae. synALDH7942 derived from Synechococcus sp. PCC7942 is a novel aldehyde dehydrogenase discovered by the present inventors, and its physiological activity has been verified in E. coli. When constructing a crocetin-producing strain using S. cerevisiae as a model, we decided to select the more suitable enzyme among the two candidates. All heterologous ALDHs were codon-optimized for S. cerevisiae and cloned into the pRS424 plasmid (Table 1). Each plasmid, pR2424-CsALDH and pRS424-synALDH7942, were transformed into the CEN-ZPC strain to construct strains CEN-ZPC-A01 and CEN-ZPC-A02 (Fig. 4A). Each engineered strain was cultured in 50 ml SD medium together with the control strain, CEN-ZPC, and the production of crocetin and the accumulation of zeaxanthin were determined using cell pellets and medium extracts. As a result, all engineered strains showed peaks with the same retention time and UV / Vis spectra as the crocetin standard (Figs. 4B and 4C). However, considering the potency (mg / L), the CEN-ZPC-A01 strain using the CsALDH enzyme was ultimately selected (Fig. 4D).

[0162]

[0163] 2.4. Confirmation of crocin-1 and crocin-2 biosynthesis using NtUGT (UDP-glycosyltransferase)

[0164] Although several UDP-glycosyltransferase enzymes that convert crocetin to crocin have been reported, the present inventors have discovered a novel enzyme, NtUGT, from Nicotiana tabacum. Among the previously discovered enzymes, the gene fragment corresponding to NtUGT, along with the gene encoding GjUGT1 from Gardenia jasminoides, were each cloned into the pRS426 plasmid and expressed in S. cerevisiae (Fig. 5A). Since standards for crocin-1 and crocin-2 are not commercially available, a medium extract of the E. coli crocin-2 strain was used as a control, and HPLC-DAD analysis was performed with an extract from S. cerevisiae. As a result, peaks with the same retention time and UV / Vis spectra as crocin-1 and crocin-2 were confirmed in both strains CEN-ZPC-A-U01 and CEN-ZPC-A-U02. Crocin-1 was confirmed in both the cell pellet and the medium together with crocetin, whereas crocin-2 was observed only in the medium (Fig. 5B, 5C). The NtUGT discovered by the present inventors exhibited the same in vivo activity as the previously reported enzyme GjUGT1, and it was confirmed that the conversion to crocin was superior when NtUGT was used (Fig. 5D). Considering the titer (mg / L), the CEN-ZPC-A-U01 strain using the NtUGT enzyme was ultimately selected.

[0165]

[0166] 2.5. Confirmation of biosynthesis of crocin-3 and crocin-4 using CaUGT3 (UDP-glycosyltransferase)

[0167] Finally, the novel enzyme CaUGT3 derived from Catharanthus roseus discovered by the inventors was cloned into the pRS426-NuUGT plasmid using the Gibson cloning method and transformed into S. cerevisiae (Fig. 6A). HPLC-DAD analysis of the medium extract of the transformed strain, CEN-ZPC-A-U1U2, detected peaks with the same retention times as crocin-3 and crocin-4 of the crocin-4-producing E. coli strain (Fig. 6B). The UV / Vis spectra (Fig. 6C) of each peak were the same as those of the crocin-3 and crocin-4 standards. In summary, we confirmed that the novel enzymes NtUGT and CaUGT3 discovered by the inventors have produced crocin-4 in S. cerevisiae for the first time in the world.

[0168]

[0169] Example 3: Construction of a biosynthetic pathway for a crocin-producing strain in S. cerevisiae.

[0170] 3.1. Integration of the CCD gene into the chromosome to construct a biosynthetic pathway for a crocetin-producing strain in S. cerevisiae.

[0171] The pPGK1_CsCCD2_tTEF2 cassette of the plasmid pRS424_CCD2 (Table 3), whose expression was confirmed by transformation, was amplified by OE-PCR by joining each fragment with primers (Table 4) containing a 50-bp homologous recombination region. The amplified cassette was integrated into the chromosome of the CEN_ZP strain using the CHOP CHOP program with a guide RNA expression vector targeting LEU2 (Table 4) and the CRISPR-Cas9 method, thereby creating the CEN_ZPEC strain. However, the peak corresponding to crocetin dialdehyde was not detected. This is presumed to be because the crocetin dialdehyde produced by CCD2 is directly converted to crocetin due to ALDH, which is endogenously expressed in S. cerevisiae.

[0172] The CsALDH gene of pRS424-CsALDH(sc) (Table 3) was amplified by OE-PCR using the CHOP CHOP program in the CEN_ZPEC strain with a guide RNA expression vector targeting HO and primers containing a 50-bp homologous recombination region (Table 4), and the pTEF1_CsADLH(sc)_tADH1 cassette was integrated into the chromosome using the CRISPR-Cas9 method, thereby constructing the CEN-ZPECA strain that produces crocetin (Fig. 7A). This strain was cultured in 50 ml SD 2X (Synthetic defined, drop out mix 2X) medium, and the production of crocetin and the accumulation of zeaxanthin were confirmed through cell pellet extracts. In the constructed CEN-ZPECA strain, a peak with the same retention time and UV / Vis spectrum as the crocetin standard was confirmed (Figs. 7B, 7C).

[0173]

[0174] 3.2. Construction of the biosynthetic pathway of a crocin-2-producing strain in S. cerevisiae

[0175] The NtUGT and GjUGT1 genes of plasmids pRS426_NtUGT, pRS426_GjUGT1 (Table 3), whose expression was confirmed by transformation, were ligated into the CEN-ZPECA strain using the CHOP CHOP program, a guide RNA expression vector targeting DPP1, and primers containing 50 bp homologous recombination regions (Table 4), and each fragment was joined by OE-PCR. The pTEF1_GjUGT1_tTEF2 cassette and pTEF1_NtUGT_tTEF2 cassette were integrated into the chromosome using the CRISPR-Cas9 method. The CEN-ZPECAG (Fig. 8A) and CEN-ZPECAN strains producing crocin-2 were constructed (Fig. 8D).

[0176] This was cultured in 50 ml SD 2X (Synthetic defined, drop out mix 2X) medium, and the production of crocin-2 and the accumulation of zeaxanthin in the cell pellet extract and medium were confirmed in CEN-ZPECAN and CEN-ZPECAG. In the produced CEN-ZPECAN and CEN-ZPECAG strains, peaks with the same retention time and UV / Vis spectra as the crocin-2 standard were confirmed (Figs. 8B, 8C, and 8E, 8F).

[0177]

[0178] 3.3. Confirmation of biosynthesis of crocin-3 and crocin-4 in S. cerevisiae using a novel enzyme, CaUGT3 (UDP-glycosyltransferase).

[0179] The pRS424_CaUGT3 plasmid (Table 3) was transformed into the CEN_ZPECAN strain (Fig. 9A). The strain was cultured in 50 ml of SD 2X (Synthetic defined, drop out mix 2X) medium, and HPLC-DAD analysis of the medium extract detected peaks with the same retention times as crocin-2 and crocin-4 of the crocin-4-producing strain of E. coli (Fig. 9B). The UV / Vis spectra of each peak were the same as those of the crocin-2 and crocin-4 standards (Fig. 9C).

[0180] Table 3. Strains and plasmids for crocetin production and crocin-2,4 production in S. cerevisiae used in this study.

[0181]

[0182]

[0183] Table 4. Oligonucleotides used in this study

[0184]

Claims

1. (a) A gene encoding tHMG1 (truncated HMG-CoA reductase); (b) gene encoding CrtE (GGPP synthase); (c) a gene encoding CrtYB (bifunctional phytoene synthase and lycopene cyclase); and (d) Recombinant yeast for producing crocin and its precursor, into which a gene encoding CrtI (phytoene desaturase) has been introduced.

2. A recombinant yeast for producing crocin and its precursor, characterized in that the recombinant yeast of claim 1 produces β-carotene, a precursor of crocin. 3.(a) Gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) a gene encoding CrtI; and (e) Recombinant yeast for producing crocin and its precursor, into which a gene encoding CrtZ (β-carotene hydroxylase) has been introduced.

4. A recombinant yeast for producing crocin and its precursor, characterized in that the CrtZ gene in claim 3 is derived from Pantoea ananatis.

5. A recombinant yeast for producing crocin and its precursor, characterized in that the recombinant yeast in claim 3 produces zeaxanthin, a precursor of crocin. 6.(a) Gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) gene encoding CrtI; (e) a gene encoding CrtZ; and (f) Recombinant yeast for producing crocin and its precursor, into which a gene encoding CCD (carotenoid cleavage dioxygenase) has been introduced.

7. A recombinant yeast for producing crocin and its precursor, characterized in that the CCD gene in paragraph 6 is derived from Crocus Sativus.

8. A recombinant yeast for producing crocin and its precursor, characterized in that the recombinant yeast in paragraph 6 produces crocetin dialdehyde, a precursor of crocin. 9.(a) Gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; and (g) Recombinant yeast for producing crocin and its precursor, into which a gene encoding ALDH (aldehyde dehydrogenase) has been introduced.

10. A recombinant yeast for producing crocin and its precursor, characterized in that the ALDH gene in claim 9 is derived from Crocus Sativus.

11. A recombinant yeast for producing crocin and its precursor, characterized in that the recombinant yeast in claim 9 produces crocetin, a precursor of crocin. 12.(a) Gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; (g) a gene encoding ALDH; and (h) Recombinant yeast for producing crocin and its precursor, into which a gene encoding NtUGT (UDP-glycosyltransferase, UDP-glycosyltransferase) derived from Nicotiana tabacum has been introduced.

13. A recombinant yeast for producing crocin and its precursors, characterized in that the recombinant yeast in claim 12 produces crocin-1 and crocin-2. 14.(a) Gene encoding tHMG1; (b) a gene encoding CrtE; (c) a gene encoding CrtYB; (d) gene encoding CrtI; (e) a gene encoding CrtZ; (f) a gene encoding CCD; (g) gene encoding ALDH; (h) a gene encoding NtUGT derived from Nicotiana tabacum; and (i) Recombinant yeast for producing crocin and its precursor, into which a gene encoding CaUGT3 (UDP-glycosyltransferase) derived from Catharanthus roseus has been introduced.

15. A recombinant yeast for producing crocin and its precursors, characterized in that the recombinant yeast in claim 14 produces crocin-3 and crocin-4.

16. A recombinant yeast for producing crocin-1 and crocin-2, wherein a gene encoding NtUGT from Nicotiana tabacum has been introduced into a yeast genetically modified to produce crocetin.

17. A recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding NtUGT derived from Nicotiana tabacum and a gene encoding CaUGT3 derived from Catharanthus roseus are introduced into a yeast genetically modified to produce crocetin.

18. A recombinant yeast for producing crocin-3 and crocin-4, wherein a gene encoding CaUGT3 derived from Catharanthus roseus has been introduced into a yeast genetically modified to produce crocin-2.

19. A recombinant yeast according to any one of claims 1 to 18, characterized in that the yeast is Saccharomyces cerevisiae.

20. A method for producing crocin and its precursor, comprising the step of culturing the recombinant yeast of any one of claims 1 to 18.

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